Molecular characterization of a deletion encompassing the splotch mutation on mouse chromosome 1

We have used a set of markers newly assigned to the proximal portion of mouse chromosome 1 to characterize the chromosomal segment deleted in the splotch-retarded (Spr) mouse mutant. Among nine markers tested in the heterozygote Spr/+mouse, we have identified four genes, Vil, Des, Inha, and Akp-3, which map within the Spr deletion. The closest distal marker to the deletion is the Acrg gene, with the distal deletion breakpoint mapping within the 0.8-cM segment separating Akp-3 and Acrg. The most proximal gene to the Spr deletion is Tp1. The proximal deletion breakpoint maps within the 0.8-cM segment separating Tp1 and Vil. The minimum size of the Spr deletion would therefore be limited to 14 cM, the genetic distance between Vil and Akp-3. The maximum size of the Spr deletion is estimated to be 16 cM, the genetic distance between Tp1 and Acrg.     

Linkage analysis of the Bcg gene on mouse chromosome 1. Identification of a tightly linked marker

We have mapped and determined the gene order of five cloned genes in the vicinity of the murine host resistance gene Bcg on mouse chromosome 1. For this, we have used a RFLP-type analysis in panels of 43 recombinant inbred strains, 3 congenic mouse strains, and 186 segregating backcross progeny derived from inbred strains of Bcgr and Bcgs genotypes. The Bcg alleles of segregating animals were established by in vivo infection with Mycobacterium bovis (Bacillus Calmette-Guérin) strain Montreal. Genomic DNA prepared from progenitor mouse strains was isolated, digested with restriction endonucleases, and analyzed by Southern blotting to identify strain-specific RFLP for each DNA marker tested. Among a number of DNA markers tested, Len2, Fn, Vil, Alpi, and Achrg were found to co-segregate with Bcg in mouse strains congenic for this locus. Detailed segregation analysis of the five markers and Bcg showed that Vil was extremely close to Bcg with no recombinant identified, whereas Fn and Len2 were located 4.5 and 9 cM proximal of Bcg, respectively. Alpi and Achrg mapped 5 and 5.5 cM distal from Bcg, respectively. Pedigree analysis in the recombinant inbred strains and backcross animals indicated the gene order: centromere-Len2-Fn-Vil,Bcg-Alpi-Achrg. The tightly linked Vil marker can now be used as an entry point in recombinant genomic DNA libraries to clone sequences overlapping Bcg. This group of five genes flanking Bcg on mouse chromosome 1 is precisely conserved on the telomeric end of the long arm of human chromosome 2q. Our results suggest that a likely location for a putative human homologue to the murine host resistance gene Bcg is the long arm of human chromosome 2 (2q32-qter).     

High-Resolution Linkage Map in the Vicinity of the Host Resistance Locus Bcg

The mouse chromosome 1 locus Bcg determines natural resistance/susceptibility of inbred mouse strains to infection with antigenically unrelated intracellular parasites, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani. In our effort to clone Bcg, we have constructed a high-resolution genetic linkage map in the vicinity of the gene. We have developed eight new highly polymorphic markers (simple sequence repeats) corresponding to cloned genes (Vil, Inha, Des), microdissected chromosome 1 anonymous probes (λMm1C136, λMm1C163, λMm1C165), or novel DNA markers from the region obtained by chromosome walking (D1Mcg101 and D1Mcg105). We have followed the cosegregation of these markers with respect to Bcg in a novel panel of 1000 (C57L/J × C57BL/6J) × C57BL/6J segregating backcross mice. Additional segregation analyses were carried out in preexisting panels of intra- and interspecific backcross mice and recombinant inbred strains. Three of these markers were found to be very tightly linked to Bcg: λMm1C165 did not show recombination with Bcg in 1424 meioses analyzed, while D1Mcg105 and λMm1C136 were located 0.1 cM proximal and 0.2 cM distal to Bcg, respectively. This analysis enabled us to define further the proximal and distal boundaries of the Bcg interval: the proximal limit was defined by a single crossover occurring between D1Mcg105 and Bcg/λMm1C165/Vil, and the distal limit by 1 crossover between Bcg/λMm1C165/Vil and λMm1C136 in 1683 and 575 informative meioses, respectively, for a maximal interval of 0.3 cM. Combined pedigree analyses for the 30-cM segment overlaping Bcg on mouse chromosome 1 indicated the locus order and the interlocus distances (in cM): centromere-Col3a1-(8.8)-Cryg-(2.6)- λMm1C163 -(1.6)-Fn-1-(2.0)-Tp-1- (1.O)-D1Mcg105-(O.1)-λMm1C165/Vil/Bcg-(O.2)-λMm1C136-(0.3)-Des/D1Mit7-(0.1)-Inha-(2.8)-λMm1C153-(2.4)-λMm1C156-(1.2)-Pax-3-(5.6)-Akp-3-(O.8)-Acrg-(2.0)-Sag-(O.5)-Col6a3.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

Genetic mapping and assignment of a long-range repeat cluster to band D of chromosome 1 in Mus musculus and M. spretus.

A cluster (D1Lub1) of a long-range repeat family was mapped to the proximal part of the Giemsa-negative band D in Chromosome 1 of Mus musculus and M. spretus by in situ hybridization with cloned probes of the long-range repeat family. By making use of restriction fragment length polymorphisms in DNAs from interspecific backcross mice, the cluster could be mapped to a position 5.3 +/- 2.1 cM distal to the Inha locus and the same distance proximal to the Bcl-2 locus. D1Lub1 was inseparable in 114 meiotic events from Acrg, Sag, and Akp-3. Taken together, the data may serve as a reference for coordinating the genetic and cytogenetic maps of Mus Chromosome 1. High-copy-number variants of the cluster, which appear cytogenetically as homogeneously staining regions at the same chromosome location, presumably arose by amplification of the long-range repeat family in situ.     

Mapping Creb-1 to chromosome 1 in the mouse.

The gene CREB1 encoding the cyclic AMP response element DNA binding protein was previously assigned to human 2q32.3-q34. In this study, a panel of 207 backcross mice made between C57BL/10ScSn (=B10) females and (B10 x B10.L-Lsh)F1 males were used to map Creb-1 with respect to Cryg and Lsh/Vil on mouse chromosome 1. A reverse-transcribed, polymerase chain reaction-amplified cDNA probe covering bp 39 to 554 of the human sequence identified restriction fragment length polymorphisms with 7/18 restriction endonucleases used to digest whole genomic mouse DNA from the parental strains. BglII and DraI RFLPs for Creb-1 were scored on a subpanel of 16/207 known recombinants between Cryg and Lsh/Vil, yielding 2/16 recombinants between Cryg and Creb-1 and 14/16 recombinants between Creb-1 and Lsh/Vil. The 16/207 recombinants observed between Lsh/Vil and Cryg provide an estimated recombination frequency of 0.077 +/- 0.019, equivalent to a map distance of 7.7 +/- 1.9 cM. This is in good agreement with previously published map distances. The number of recombinants observed between Creb-1 and the other markers place Creb-1 approximately 1 cM distal to Cryg and 7 cM proximal to Lsh/Vil.     

The L-isoaspartyl/D-aspartyl protein methyltransferase gene (PCMT1) maps to human chromosome 6q22.3-6q24 and the syntenic region of mouse chromosome 10.

We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) using cDNA probes. We determined that the human gene is present in chromosome 6 by Southern blot analysis of DNA from a panel of mouse-human somatic cell hybrids. In situ hybridization studies allowed us to confirm this identification and further localize the human gene (PCMT1) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we localized the mouse gene (Pcmt-1) to chromosome 10, at a position 8.2 +/- 3.5 cM proximal to the Myb locus. This region of the mouse chromosome is homologous to the human 6q24 region.     

Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.     

Localization of the rhodopsin gene to the distal half of mouse chromosome 6

We have assigned the mouse rhodopsin gene, Rho, to chromosome 6 using DNA from a set of mouse-hamster somatic hybrid cell lines and a partial cDNA clone for mouse opsin. This assignment rules out the direct involvement of the rhodopsin gene in the known mouse mutations that produce retinal degeneration, including retinal degeneration slow (rds, chromosome 17), retinal degeneration (rd, chromosome 5), Purkinje cell degeneration (pcd, chromosome 13), and nervous (nr, chromosome 8). Segregation of Rho-specific DNA fragment differences among 50 animals from an interspecific backcross (C57BL/6J X Mus spretus) X C57BL/6J indicates that the Rho locus is 4.0 +/- 2.8 map units distal to the locus for the proto-oncogene Raf-1 and 18.0 +/- 5.4 map units proximal to the locus for the proto-oncogene Kras-2. Linkage to Raf-1 was confirmed using four sets of recombinant inbred strains. The two loci RAF1 and RHO are also syntenic on human chromosome 3, but on opposite arms.     

A high-resolution radiation hybrid map of the proximal portion of mouse chromosome 5

Radiation hybrid (RH) mapping of the mouse genome provides a useful tool in the integration of existing genetic and physical maps, as well as in the ongoing effort to generate a dense map of expressed sequence tags. To facilitate functional analysis of mouse Chromosome 5, we have constructed a high-resolution RH map spanning 75 cM of the chromosome. During the course of these studies, we have developed RHBase, an RH data management program that provides data storage and an interface to several RH mapping programs and databases. We have typed 95 markers on the T31 RH panel and generated an integrated map, pooling data from several sources. The integrated RH map ranges from the most proximal marker, D5Mit331 (Chromosome Committee offset, 3 cM), to D5Mit326, 74.5 cM distal on our genetic map (Chromosome Committee offset, 80 cM), and consists of 138 markers, including 89 simple sequence length polymorphic markers, 11 sequence-tagged sites generated from BAC end sequence, and 38 gene loci, and represents average coverage of approximately one locus per 0.5 cM with some regions more densely mapped. In addition to the RH mapping of markers and genes previously localized on mouse Chromosome 5, this RH map places the alpha-4 GABA(A) receptor subunit gene (Gabra4) in the central portion of the chromosome, in the vicinity of the cluster of three other GABA(A) receptor subunit genes (Gabrg1-Gabra2-Gabrb1). Our mapping effort has also defined a new cluster of four genes in the semaphorin gene family (Sema3a, Sema3c, Sema3d, and Sema3e) and the Wolfram syndrome gene (Wfs1) in this region of the chromosome.     

Mapping of Col3a1 and Col6a3 to proximal murine chromosome 1 identifies conserved linkage of structural protein genes between murine chromosome 1 and human chromosome 2q

We have investigated the degree of synteny between the long arm (q) of human chromosome 2 and the proximal portion of mouse chromosome 1. To define the limits of synteny, we have determined whether mouse homologs of seven human genes mapping to chromosome 2q cosegregated with anchor loci on mouse chromosome 1. The loci investigated were NEB/Neb, ELN/Eln, COL3A1/Col3a1, CRYG/Len-2, FN1/Fn-1, VIL/Vil, and COL6A3/Col6a3. Ren-1,2 and Acrg were included as two proximal mouse chromosome 1 anchor loci. The segregation of restriction fragment length polymorphisms at these loci was analyzed in the progeny of Mus spretus x C57BL/6J hybrids backcrossed to the C57BL/6J inbred strain. We found that five of the structural protein loci and the two anchor loci form a linkage group on proximal murine chromosome 1. The proposed gene order of this group of linked markers is centromere - Col3a1 - Len-2-Fn-1-Vil-Acrg-Col6a3-Ren1,2. Neb and Eln are linked neither to each other nor to any other marker on proximal mouse chromosome 1. Therefore, the mouse loci Col3a1 and Col6a3 are identified as flanking markers of the linkage group of structural protein loci. The estimated genetic map distances are Col3a1-13.3 cM-Len-2-3.4 cM-Fn-1-3.8 cM-Vil-9.6 cM-Acrg-2.1 cM-Col6a3-18.3 cM-Ren1,2. The available map information for human chromosome 2q markers and mouse chromosome 1 markers presented here tentatively identifies Col3a1 and Col6a3 as the border markers that define the limits of the syntenic chromosome segment. The order of mouse genes on chromosome 1 and their human homologs on chromosome 2q also appears to be conserved, suggesting that mapping of murine genes on the conserved segment may be useful to predict gene order in man.     

Analyses of beta-1 syntrophin, syndecan 2 and gem GTPase as candidates for chicken muscular dystrophy.

Despite intensive studies of muscular dystrophy of chicken, the responsible gene has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to chromosome 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11-24.3, where the beta-1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) genes are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease.     

A glutaminase (gis) gene maps to mouse chromosome 1, rat chromosome 9, and human chromosome 2

A rat cDNA clone encoding a portion of phosphate-activated glutaminase was used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between wild-derived and inbred strains of mice. Segregation of rat and mouse chromosomes among somatic cell hybrids indicated assignment to rat chromosome 9 and mouse chromosome 1. Analysis of chromosome 1 alleles for several genes in an interspecific cross between Mus spretus and C3H/HeJ-gld/gld mice indicates that glutaminase can be positioned within 5.5 +/- 2.0 cM proximal to Ctla-4. Similarly, human-hamster somatic cell hybrids were examined for RFLPs, and four human EcoRI restriction fragments were found to hybridize with the rat glutaminase probe. Two of these restriction fragments cosegregated and mapped to human chromosome 2 in a region that is syntenic with mouse chromosome 1 and rat chromosome 9.     

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase beta-subunit gene in mouse and human: tight linkage to the Huntington disease region (4p16.3).

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE beta-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 +/- 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.     

Mitochondrial malate dehydrogenase (Mor-1) in the mouse: Linkage to chromosome 5 markers

Malate dehydrogenase is present in most mammalian tissues in both supernatant and mitochondrial forms. Although genetic variation for the supernatant form has not been observed in the mouse, electrophoretic variants caused by alleles at the mitochondrial locus (Mor-1) have been previously described. We have located this locus 11.0 +/- 2.9 cM from the beta-glucuronidase structural gene, Gus, on chromosome 5. The gene order is Hm-Pgm-1-rd-bf-Gus-Mor-1. Thus Mor-1 is presently the most distal marker on chromosome 5. Three different nuclear loci for mitochondrial enzymes (Mod-2, Got-2, and Mor-1) have now been mapped in the mouse, all on different chromosomes.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

The mouse IFN-alpha (Ifa) locus: correlation of physical and linkage maps by in situ hybridization

The physical location of the mouse IFN-alpha locus (Ifa) on chromosome 4 was defined by in situ hybridization of a cloned mouse IFN-alpha probe to metaphase spreads in which one chromosome 4 was present as part of a single metacentric chromosome, all other chromosomes being acrocentric. (This approach greatly facilitates analysis and can be used even when it is difficult to obtain good banding). Using unbanded chromosomes, the grains were localized over the chromosome 4 part of the metacentric, in a region 0.61 +/- 0.07 (SD) of the distance from the centromere to the telomere. In Giemsa-banded spreads, the majority of the grains were in the region 4C3----C6. Consideration of these results and of the known linkage maps for mouse and man indicates that the Galt - Aco-1 - Ifa syntenic group spans a distance of approximately 14 cM and suggests that the same group on human 9p will also occupy a similarly sized region, with GALT proximal and IFL distal to the centromere.     

Genetic and physical maps of jerker (Espn(je)) on mouse chromosome 4

The jerker mutation causes degeneration of cochlea and vestibular sensory hair cells in mice. A frame-shift mutation in the actin bundling gene Espin (Espn) leads to hair bundle defects by disrupting the actin filament assembly in stereocilia. Previously, jerker was mapped to distal mouse chromosome 4. Here, analyzing 2536 informative meioses derived from two intersubspecific intercrosses, we localize jerker to a 0.51+/-0.14cM interval on chromosome 4. The following order and distances of genes and markers were determined: D4Mit180-0.44+/-0.13cM-Hes2, Espn(je)-0.08+/-0.06cM-D4Mit356-0.28+/-0.1cM-D4Mit208. A 300kb physical bacterial artificial chromosome (BAC) contig was generated containing the Espn(je) locus. The human homologous region maps to 1p36.31. We present a detailed high-resolution genetic and physical map of markers located at distal chromosome 4 and demonstrate concordance of Espn with jerker.